Histone methylation and demethylation are implicated in the transient and sustained activation of the interleukin-1ß gene in murine macrophages.

Macrophages play a crucial role in the development of atherosclerosis. To explore the mechanism by which macrophages attain a proinflammatory phenotype for a sustained period, we stimulated macrophages with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) and measured the interleukin-1ß (IL-1ß) expression. The IL-1ß expression increased transiently, and its expression lasted for, at least, 1 week after the cessation of LPS and IFN-γ stimulation. At the promoter region of the IL-1ß gene, the demethylation of histone H3 lysine 27 (H3K27) was significantly induced for 1 week after transient stimulation with LPS and IFN-γ. The expression of H3K27 demethylases ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX) and jumonji domain-containing 3 (JMJD3) increased significantly for 1 week after transient stimulation with LPS and IFN-γ. When the UTX expression was inhibited by using small interfering RNA (siRNA) for UTX, the IL-1ß expression was significantly suppressed in both transient and sustained phases, whereas siRNA for JMJD3 significantly inhibited only the sustained phase of the IL-1ß expression. These results suggested that H3K27 demethylation was implicated in the transient and sustained increase in the IL-1ß expression after LPS and IFN-γ stimulation.

Endogenous Interleukin-1ß Is Implicated in Intraplaque Hemorrhage in Apolipoprotein E Gene Null Mice.

BACKGROUND: Intraplaque hemorrhage (IPH) has been implicated in plaque instability and rupture in atherosclerotic lesions, although the mechanisms by which IPH progresses remain largely unknown. In this study, apolipoprotein E-deficient mice with carotid artery ligation and cuff placement around the artery were used, and pro-inflammatory cytokines that are implicated in IPH were analyzed.Methods and Results:The expression of interleukin-1ß (IL-1ß) increased significantly following cuff placement compared with mice with carotid artery ligation alone. IPH occurred in the cuff-placed carotid artery following treatment with the negative control (NC) small interfering RNA (siRNA). However, the occurrence was significantly reduced in the cuff-placed carotid artery following treatment with an IL-1ß siRNA. Neovessel formation was significantly reduced in the carotid artery treated with the NC siRNA compared with that treated with IL-1ß siRNA. IL-1ß significantly inhibited the tube formation and wound healing capacities of vascular endothelial cells in vitro. Furthermore, immunostaining of matrix metalloproteinase-9 (MMP-9) significantly increased in the carotid artery treated with the NC siRNA compared with that treated with IL-1ß siRNA. CONCLUSIONS: These results suggest that endogenous IL-1ß is implicated in the progression of IPH via the inhibition of physiological angiogenesis in the atherosclerotic plaque, leading to the formation of leaky neovessels. Furthermore, the stimulation of MMP-9 expression may also contribute to the formation of leaky neovessels.

Therapeutic Effects of Mesenchymal Stem Cell-Derived Exosomes in Cardiovascular Disease.

Mesenchymal stem cells (MSCs) are multipotent stem cells that reside in various organs. They have the capacity to differentiate into various cell types, including cardiomyocytes, vascular endothelial cells, and vascular smooth muscle cells. Among the various MSCs, bone marrow-derived MSCs (BMMSCs) have been widely used for treating acute myocardial infarction (AMI) and ischemic heart failure (IHF) in preclinical and clinical studies. Although the beneficial effects of BMMSCs in treating AMI and IHF were originally attributed to their capacity to differentiate into cardiac cell types, recent evidence suggests that the differentiation capacity of BMMSCs appears to be minimal and that BMMSCs exert cardioprotective effects by secreting paracrine factors. In this context, MSC-derived exosomes have recently gained much attention. In this chapter, we introduce preclinical studies in which MSC-derived exosomes are used for treating cardiovascular diseases (CVDs) such as AMI, stroke, pulmonary hypertension, and septic cardiomyopathy. Future clinical studies are required to confirm the efficacy of exosome administration in treating CVDs.

Influence of In Vitro Hemolysis on 80 Different Laboratory Tests.

BACKGROUND: In vitro hemolysis is probably the most common pre-analytic problem in laboratory medicine. However, it introduces variation into results in unknown ways. Therefore, the purpose of this study was to assess the quantitative effects of hemolysis on 80 different, routine laboratory tests. METHODS: We examined the ratio of hemolysis in our hospital from January 1 to March 31, 2015. Next, to study the effect of in vitro hemolysis of whole blood, we added lysed erythrocytes to pooled specimens of serum or plasma to give hemoglobin concentrations of 0.9 to 8.1 g/L and 2.8 to 14 g/L, respectively, and a rating by colorimetry of 0 to 4+ hemolyzed. Then, 80 different laboratory tests were determined with a Hitachi 7700 autoanalyzer for biochemical tests and with AIA 2000, Cobas 6000, and Lumipulus G1200 machines for other tests. RESULTS: Hemolysis occurred in a total in 8.6% of the specimens in our hospital. Significant correlations with the hemolysis ratio were observed in 43 of 80 laboratory tests. At apparent hemolysis, 11 test levels increased and 7 test levels decreased due to hemolysis. Among the 11 tests, potassium, aspartate aminotransferase, lactate dehydrogenase, thymol turbidity test (TTT), zinc sulfate turbidity test (ZTT), and hyaluronic acid tests showed proportional increases due to hemolysis. CONCLUSIONS: Hemolysis is a common problem for accuracy in many routine laboratory tests. Although the quantitative effects of hemolysis can only be roughly estimated in this report, the approximate extent of change in specific laboratory tests is useful for establishing a baseline for future hemolytic studies.

Stem cell-derived exosomes as a therapeutic tool for cardiovascular disease.

Mesenchymal stem cells (MSCs) have been used to treat patients suffering from acute myocardial infarction (AMI) and subsequent heart failure. Although it was originally assumed that MSCs differentiated into heart cells such as cardiomyocytes, recent evidence suggests that the differentiation capacity of MSCs is minimal and that injected MSCs restore cardiac function via the secretion of paracrine factors. MSCs secrete paracrine factors in not only naked forms but also membrane vesicles including exosomes containing bioactive substances such as proteins, messenger RNAs, and microRNAs. Although the details remain unclear, these bioactive molecules are selectively sorted in exosomes that are then released from donor cells in a regulated manner. Furthermore, exosomes are specifically internalized by recipient cells via ligand-receptor interactions. Thus, exosomes are promising natural vehicles that stably and specifically transport bioactive molecules to recipient cells. Indeed, stem cell-derived exosomes have been successfully used to treat cardiovascular disease (CVD), such as AMI, stroke, and pulmonary hypertension, in animal models, and their efficacy has been demonstrated. Therefore, exosome administration may be a promising strategy for the treatment of CVD. Furthermore, modifications of exosomal contents may enhance their therapeutic effects. Future clinical studies are required to confirm the efficacy of exosome treatment for CVD.

The angiotensin II receptor antagonist, losartan, enhances regulator of G protein signaling 2 mRNA expression in vascular smooth muscle cells of Wistar rats.

Angiotensin II (Ang II) reportedly enhances regulator of G-protein signaling 2 (RGS2), thus making a negative feedback loop for Ang II signal transduction. However, few studies have reported whether Ang II receptor (ATR) antagonists influence RGS2 mRNA expression. We investigated RGS2 mRNA expression when Ang II binding to ATR was blocked with Ang II subtype-1 receptor (AT1R) blockers using vascular smooth muscle cells from the thoracic aorta of male Wistar rats. RGS2 mRNA expression significantly increased with Ang II stimulation, and this increase was almost completely abolished by olmesartan, a potent AT1R-specific blocker. Ang II subtype-2 receptor (AT2R) was not involved in Ang II-mediated RGS expression. In contrast, the AT1R blocker, losartan, partially decreased Ang II-mediated RGS2 mRNA expression because this antagonist directly stimulated RGS2 mRNA expression in Ang II-free medium. EXP3174, which is an active metabolite of losartan, almost completely blunted Ang II-mediated RGS2 mRNA expression without direct stimulation of RGS2 mRNA expression. Moreover, pretreatment with olmesartan abolished Ang II-mediated RGS2 mRNA expression. Treatment with a protein kinase C inhibitor partially decreased losartan-mediated RGS2 mRNA expression. These results suggest that AT1R blockers inhibit RGS2 mRNA expression in response to Ang II via an AT1R-mediated mechanism. However, the AT1R blocker, losartan, behaves as a direct agonist for RGS2 mRNA expression via AT1R through protein kinase C-dependent and -independent pathways. In conclusion, losartan exhibits dual effects on RGS2 mRNA expression, and the direct upregulation of RGS2 mRNA expression may provide a new strategy for the treatment of hypertension.

Adult stem cells as a tool for kidney regeneration.

Kidney regeneration is a challenging but promising strategy aimed at reducing the progression to end-stage renal disease (ESRD) and improving the quality of life of patients with ESRD. Adult stem cells are multipotent stem cells that reside in various tissues, such as bone marrow and adipose tissue. Although intensive studies to isolate kidney stem/progenitor cells from the adult kidney have been performed, it remains controversial whether stem/progenitor cells actually exist in the mammalian adult kidney. The efficacy of mesenchymal stem cells (MSCs) in the recovery of kidney function has been demonstrated in animal nephropathy models, such as acute tubular injury, glomerulonephritis, renal artery stenosis, and remnant kidney. However, their beneficial effects seem to be mediated largely via their paracrine effects rather than their direct differentiation into renal parenchymal cells. MSCs not only secrete bioactive molecules directly into the circulation, but they also release various molecules, such as proteins, mRNA, and microRNA, in membrane-covered vesicles. A detailed analysis of these molecules and an exploration of the optimal combination of these molecules will enable the treatment of patients with kidney disease without using stem cells. Another option for the treatment of patients with kidney disease using adult somatic cells is a direct/indirect reprogramming of adult somatic cells into kidney stem/progenitor cells. Although many hurdles still need to be overcome, this strategy will enable bona fide kidney regeneration rather than kidney repair using remnant renal parenchymal cells.

Impaired response of regulator of Gαq signaling-2 mRNA to angiotensin II and hypertensive renal injury in Dahl salt-sensitive rats.

Dahl salt-sensitive (Dahl S) rats are prone to salt-dependent hypertension with severe organ damage, including stroke, cardiac failure and renal insufficiency. The mechanism for this susceptibility to kidney injury has not been elucidated. The present study proposed that an upregulation of intracellular signaling of angiotensin II (Ang-II) is responsible for the susceptibility to hypertensive kidney injury in Dahl S rats. Spontaneously hypertensive rats exhibited higher systolic blood pressure (SBP) and lower kidney damage than Dahl S rats fed a high-salt diet for 2 weeks. Ang-II infusion for 4 weeks significantly increased SBP in Dahl S and Dahl salt-resistant (Dahl R) rats fed a low-salt diet. The increase in SBP in Dahl S rats was associated with significant kidney injury with greater glomerular sclerosis (P<0.001). The expression of regulatory protein of Gαq signaling-2 (RGS2) mRNA in the aortic walls in response to Ang-II infusion was lower in Dahl S than Dahl R rats (P<0.05). Ang-II significantly increased RGS2 mRNA in the aorta in Dahl R rats, but the response was apparently blunted in Dahl S rats. These results suggest that Dahl S rats exhibit a blunted RGS2 response to Ang-II, and this blunted response may be partially responsible for the susceptibility to renal injury in Dahl S rats.

Adipose tissue-derived stem cells as a therapeutic tool for cardiovascular disease.

Adipose tissue-derived stem cells (ADSCs) are adult stem cells that can be easily harvested from subcutaneous adipose tissue. Many studies have demonstrated that ADSCs differentiate into vascular endothelial cells (VECs), vascular smooth muscle cells (VSMCs), and cardiomyocytes in vitro and in vivo. However, ADSCs may fuse with tissue-resident cells and obtain the corresponding characteristics of those cells. If fusion occurs, ADSCs may express markers of VECs, VSMCs, and cardiomyocytes without direct differentiation into these cell types. ADSCs also produce a variety of paracrine factors such as vascular endothelial growth factor, hepatocyte growth factor, and insulin-like growth factor-1 that have proangiogenic and/or antiapoptotic activities. Thus, ADSCs have the potential to regenerate the cardiovascular system via direct differentiation into VECs, VSMCs, and cardiomyocytes, fusion with tissue-resident cells, and the production of paracrine factors. Numerous animal studies have demonstrated the efficacy of ADSC implantation in the treatment of acute myocardial infarction (AMI), ischemic cardiomyopathy (ICM), dilated cardiomyopathy, hindlimb ischemia, and stroke. Clinical studies regarding the use of autologous ADSCs for treating patients with AMI and ICM have recently been initiated. ADSC implantation has been reported as safe and effective so far. Therefore, ADSCs appear to be useful for the treatment of cardiovascular disease. However, the tumorigenic potential of ADSCs requires careful evaluation before their safe clinical application.

Neuromedin B Restores Erectile Function by Protecting the Cavernous Body and the Nitrergic Nerves from Injury in a Diabetic Rat Model.

Erectile dysfunction (ED) is a major health problem worldwide and affects approximately 75% of diabetic patients, likely due to severely damaged cavernous body. While screening for cytokines produced by adipose tissue-derived stem cells, we detected neuromedin B (NMB). To explore a potential treatment option for ED, we examined whether NMB was capable of restoring erectile function. We also examined the potential mechanism by which NMB could restore erectile function. Male Wistar rats were injected with streptozotocin (STZ) to induce diabetes. An adenovirus expressing NMB (AdNMB) was injected into the penis 6 weeks after STZ administration. Four weeks after the injection of AdNMB, erectile function, penile histology, and protein expression were analyzed. As assessed by the measurement of intracavernous pressure, AdNMB injection significantly restored erectile function compared with the injection of an adenovirus expressing green fluorescent protein. This restoration was associated with conservation of the cavernous body structure and neural nitric oxide synthase (nNOS)-expressing nerves, together with recovery of α-smooth muscle actin, vascular endothelial-cadherin, and nNOS expression. Furthermore, NMB significantly stimulated the survival of SH-SY5Y cells derived from human neuroblastoma tissue with characteristics similar to neurons. Collectively, these results suggested that NMB restored erectile function via protection of the cavernous body from injury and stimulation of the survival of the associated nerves. NMB may be useful to treat ED patients with a severely damaged cavernous body.

Senescent Cells Impair Erectile Function through Induction of Endothelial Dysfunction and Nerve Injury in Mice.

Erectile dysfunction (ED) is a major health problem, particularly in the elderly population, which is rapidly increasing. It is necessary to elucidate the mechanism by which ED occurs in the elderly. Cellular senescence is commonly detected in old tissues, and it is well known that senescent cells not only withdraw from the cell cycle but also remain viable and actively produce a variety of cytokines. We examined the effect of senescent cells on erectile function after injection of senescent cells into the penises of mice. Human umbilical vein endothelial cells were infected with an adenovirus expressing a constitutively active mutant of Ras to induce senescence, and were injected into the penises of nude mice. These senescent cells expressed proinflammatory cytokines such as interleukin-1ß (IL-1ß). Injection of senescent cells impaired erectile function, as assessed by the measurement of intracavernous pressure. Although the structure of the cavernous body did not remarkably change, expression of the catalytically active form of endothelial nitric oxide synthase and that of total neural nitric oxide synthase significantly decreased after injection. The penises injected with the senescent cells expressed human IL-1ß and subsequently endogenous proinflammatory cytokines such as mouse IL-1ß and tumor necrosis factor-α. These results suggested that senescent cells impaired erectile function through induction of endothelial dysfunction and nerve injury. These effects may be mediated by proinflammatory cytokines produced by senescent cells.

Chronic kidney disease and erectile dysfunction.

Erectile dysfunction (ED) is a common condition among male chronic kidney disease (CKD) patients. Its prevalence is estimated to be approximately 80% among these patients. It has been well established that the production of nitric oxide from the cavernous nerve and vascular endothelium and the subsequent production of cyclic GMP are critically important in initiating and maintaining erection. Factors affecting these pathways can induce ED. The etiology of ED in CKD patients is multifactorial. Factors including abnormalities in gonadal-pituitary system, disturbance in autonomic nervous system, endothelial dysfunction, anemia (and erythropoietin deficiency), secondary hyperparathyroidism, drugs, zinc deficiency, and psychological problems are implicated in the occurrence of ED. An improvement of general conditions is the first step of treatment. Sufficient dialysis and adequate nutritional intake are necessary. In addition, control of anemia and secondary hyperparathyroidism is required. Changes of drugs that potentially affect erectile function may be necessary. Further, zinc supplementation may be necessary when zinc deficiency is suspected. Phosphodiesterase type 5 inhibitors (PDE5Is) are commonly used for treating ED in CKD patients, and their efficacy was confirmed by many studies. Testosterone replacement therapy in addition to PDE5Is may be useful, particularly for CKD patients with hypogonadism. Renal transplantation may restore erectile function. ED is an early marker of cardiovascular disease (CVD), which it frequently precedes; therefore, it is crucial to examine the presence of ED in CKD patients not only for the improvement of the quality of life but also for the prevention of CVD attack.

Adipose-derived stem cells stimulate reendothelialization in stented rat abdominal aorta.

BACKGROUND: Although drug-eluting stents (DES) have been widely used for the treatment of coronary artery disease, they potentially increase the risk of late thrombosis. It is, therefore, desirable to establish a strategy to stimulate reendothelialization. Endothelial injury models have been widely used to analyze the mechanisms of coronary restenosis. However, animal models deployed with coronary stents in the blood vessels are necessary to accurately analyze the mechanisms of coronary restenosis and late thrombosis because persistent inflammation occurs around the coronary stents. METHODS AND RESULTS: Coronary stents were implanted into rat abdominal aorta and adipose tissue-derived stem cells (ASC) were administered from the adventitial side. Reendothelialization was then visualized by Evans blue staining, and neointimal formation was analyzed histologically. ASC significantly stimulated reendothelialization and inhibited neointimal formation in bare metal stents (BMS)-implanted aorta. In addition, ASC promoted reendothelialization in DES-implanted aorta; however, the effects were weaker than in BMS-implanted aorta. Among the cytokines that ASC produce, adrenomedullin (AM) significantly stimulated reendothelialization and inhibited neointimal formation in BMS-implanted aorta, when an adenovirus expressing AM was administered from the adventitial side. CONCLUSIONS: These results suggest that ASC produce several cytokines that stimulate reendothelialization and inhibit neointimal formation in stent-deployed vessels, and that AM could mediate these effects.

A useful method of identifying of miRNAs which can down-regulate Zeb-2.

BACKGROUND: Although identification of the target mRNAs of micro RNAs (miRNAs) is essential to understanding their function, the low complementarity between miRNAs and their target mRNAs has complicated this process. In this study, we sought to identify miRNAs which reduce the expression of the transcription factor Zeb-2, a transcriptional repressor of E-cadherin which is known to be down regulated by members of the miR-200 family (miR-200a,b,c miR-429, and miR-141). FINDINGS: We first used a computational target predicting system to identify 82 candidate miRNAs which bound the 3'UTR region of the Zeb-2 mRNA. Of these 82 miRNAs, precursors for 51 were available in our miRNA precursor library. Pre-miR™ Precursor Molecules for these 51 miRNAs were co-transfected into NIH3T3 cells with a luciferase reporter vector containing the 3'UTR region of the Zeb-2 mRNA. Seven miRNAs (miR-141, mi-183, miR-200a, miR-200b, miR-200c, miR-429 and miR-666-5p) were shown to down-regulate luciferase activity and Western blotting analysis confirmed that Pre-miR™ Precursor Molecules for these seven miRNAs induced expression of E-cadherin and miScript target protector against miR-183 and miR-666-5p abrogated this effect. Moreover, an Anti-miR™ miRNA Inhibitor targeting miR-183 and miR-666-5p repressed expression of E-cadherin. CONCLUSIONS: We have established a method to identify miRNAs that bind the 3'UTR region of the Zeb-2 mRNA and that induce expression of E-cadherin, possibly by down-regulating the expression of Zeb-2. Our method may be more widely applicable for identifying miRNAs that bind target mRNA 3'UTR regions and down-regulate the expression of proteins encoded by these mRNAs.

Oncogene- and oxidative stress-induced cellular senescence shows distinct expression patterns of proinflammatory cytokines in vascular endothelial cells.

Senescent cells are metabolically active and produce a variety of proinflammatory cytokines. It was previously reported that atherosclerotic plaques contain senescent cells, suggesting that senescence may contribute to the progression of atherosclerosis. In this study, we induced cellular senescence in vascular endothelial cells (VECs) using hydrogen peroxide (H2O2) or an adenovirus that expresses a constitutively active mutant of Ras (AdRas12V) and studied the expression of cytokines. Both H2O2 treatment and AdRas12V infection induced senescence in VECs, as assessed by senescence-associated ß-Gal activity and the expression of proteins such as p53 and p21(CIP1). In addition, both treatments induced the expression of a variety of cytokines, including interleukin-1ß (IL-1ß) and nerve growth factor (NGF). AdRas12V infection induced IL-1ß expression more significantly than H2O2 treatment, whereas both treatments induced comparable mRNA and protein expression levels of NGF. These results suggest that senescent cells express different patterns of proinflammatory cytokines, depending on the trigger that induced senescence. It is therefore possible that senescent cells can differentially induce inflammation in the surrounding tissues, depending on the cause of senescence.

Adrenomedullin and angiopoietin-1 additively restore erectile function in diabetic rats: comparison with the combination therapy of vascular endothelial growth factor and angiopoietin-1.

INTRODUCTION: Erectile dysfunction (ED) is a major health problem. We have shown that adrenomedullin (AM) restores erectile function in diabetic rats. AIM: The aim of this study is to explore a better treatment for ED, we examined whether combination of AM and angiopoietin-1 (Ang-1) was more effective to treat ED than treatment with AM alone or Ang-1 alone. We also compared the effect of the combination therapy with that of treatment with vascular endothelial growth factor-A (VEGF-A). METHODS: Male Wistar rats were injected with streptozotocin (STZ) to induce diabetes. Adenoviruses expressing AM (AdAM), Ang-1 (AdAng-1), and VEGF-A (AdVEGF-A) were injected into the penis 6 weeks after STZ administration. Erectile function, penile histology, and protein expression were analyzed 4 weeks after the injection of the adenoviruses. MAIN OUTCOME MEASURES: Intracavernous pressure and mean arterial pressure were measured to evaluate erectile function. The morphology of the penis was analyzed by Elastica van Gieson stain and immunohistochemistry. The expression of α-smooth muscle actin (SMA), VE-cadherin and type I collagen was assessed by Western blot analysis. RESULTS: Infection with AdAM plus AdAng-1 more effectively restored erectile function than infection with AdAM alone or AdAng-1 alone. This combination therapy restored erectile function to a level similar to that observed in the age-matched Wistar rats. Expression of SMA and VE-cadherin increased more significantly in the AdAM plus AdAng-1-treated group than in the AdAM- or AdAng-1-treated group. Although AdVEGF-A infection restored erectile function significantly, it also caused enlargement of the trabeculae of the cavernous body, aberrant angiogenesis, and overproduction of type I collagen. CONCLUSIONS: These results suggested that combination therapy with AM and Ang-1 potently restored erectile function and normal morphology of the cavernous body compared with VEGF-A administration. This combination therapy will be useful to treat ED patients with a severely damaged cavernous body.

Angiopoietin-1 mediates adipose tissue-derived stem cell-induced inhibition of neointimal formation in rat femoral artery.

BACKGROUND: Adipose tissue-derived stem cells (ASC) produce a variety of cytokines that potentially mediate the proangiogenic and antiapoptotic effects of the ASC. We examined whether ASC produced angiopoietin-1 (Ang1) and whether Ang1 functionally mediated ASC-induced suppression of neointimal formation. METHODS AND RESULTS: Ang1 production was measured by enzyme-linked immunosorbent assay. Production of endogenous Ang1 by ASC was inhibited with small interfering RNA (siRNA) for Ang1. Overproduction of Ang1 was achieved with an adenovirus that expresses Ang1 (AdAng1). ASC expressing Ang1 siRNA, or AdAng1 were administered around the femoral artery after wire injury, and immunohistochemical analysis was performed to examine their effects on neointimal formation. ASC produced Ang1 in a time-dependent manner, especially when cultured in medium containing growth factors for vascular endothelial cells. When ASC were treated with Ang1 siRNA, the inhibitory effect of ASC on neointimal formation was significantly reduced. Knockdown of Ang1 significantly increased macrophage infiltration in the neointima, and significantly decreased endothelial regeneration. In contrast, forced expression of Ang1 using AdAng1 significantly suppressed neointimal formation and macrophage infiltration, and stimulated reendothelialization. CONCLUSIONS: Ang1 was implicated in ASC-induced suppression of neointimal formation. The results also suggested that Ang1 inhibited neointimal formation via stimulation of reendothelialization and suppression of macrophage infiltration in the neointima.

Adrenomedullin mediates adipose tissue-derived stem cell-induced restoration of erectile function in diabetic rats.

INTRODUCTION: Erectile dysfunction (ED) is a major health problem. It is known that diabetic patients are more refractory to common treatments for ED. AIM: To explore the better treatment for ED, we examined the effects of adipose-derived stem cells (ASC) on ED using a diabetic rat model. We also analyzed the cytokines produced by ASC and implicated in ASC-induced restoration of erectile function. METHODS: Male Wistar rats were injected with streptozotocin (STZ) to induce diabetes. ASC or adenoviruses were injected into the penis 6 weeks after STZ administration. Erectile function, penile histology and protein expression were analyzed 4 weeks after the injection of ASC or adenoviruses. MAIN OUTCOME MEASURES: Intracavernous pressure and mean arterial pressure were measured to evaluate erectile function. The morphology of the penis was analyzed by Elastica van Gieson stain and immunohistochemistry. The expression of proteins specific for vascular endothelial cells (VEC) was assessed by Western blot analysis. RESULTS: ASC restored erectile function especially when they were cultured in medium containing growth factors for VEC. This restoration was associated with improvement in the histology of the cavernous body, and increased expression of VEC markers such as VE-cadherin and endothelial nitric oxide synthase (eNOS). When the expression of adrenomedullin (AM), a vasoactive peptide originally isolated from human pheochromocytoma tissue, was knocked down, the effect of ASC on ED was significantly diminished. Knockdown of AM was associated with decreased expressions of VE-cadherin and eNOS. Furthermore, overexpression of AM induced by adenovirus infection significantly improved erectile function in these diabetic rats. Overexpression of AM was associated with increased expressions of VE-cadherin and eNOS. CONCLUSIONS: These results suggested that ASC have the potentials to restore erectile function and that AM produced by ASC plays a major role in the restoration of erectile function.

Renoprotective effect of erythropoietin in ischemia/reperfusion injury: possible roles of the Akt/endothelial nitric oxide synthase-dependent pathway.

OBJECTIVES: It has been reported that erythropoietin protects the kidneys from ischemia/reperfusion injury. In the present study, we examined the role of Akt and endothelial nitric oxide synthase in the protective effect of erythropoietin on ischemia/reperfusion injury of the kidney. METHODS: Erythropoietin was injected in the peritoneal space of ICR mice after ischemia/reperfusion injury and its effect was assessed by measuring blood urea nitrogen and creatinine, and by histological analysis. Phosphorylation of Akt and endothelial nitric oxide synthase was examined by western blot analysis. Endothelial nitric oxide synthase gene null mice were also used to examine the role of endothelial nitric oxide synthase in the renoprotective effect of erythropoietin. RESULTS: Erythropoietin administration significantly inhibited the increase in blood urea nitrogen and creatinine after ischemia/reperfusion injury compared with control mice. Accordingly, erythropoietin administration significantly ameliorated the histological damages, including apoptotic cell death. Erythropoietin significantly stimulated phosphorylation of Akt and endothelial nitric oxide synthase in the kidneys. When endothelial nitric oxide synthase gene null mice were subjected to ischemia/reperfusion injury, erythropoietin did not significantly suppress the increase in blood urea nitrogen or creatinine. CONCLUSIONS: Erythropoietin seems to activate the Akt/endothelial nitric oxide synthase-dependent pathway in the kidneys. This pathway might be implicated in the renoprotective effect of erythropoietin in the ischemia/reperfusion injury model.

miR-200b precursor can ameliorate renal tubulointerstitial fibrosis.

Members of the miR-200 family of micro RNAs (miRNAs) have been shown to inhibit epithelial-mesenchymal transition (EMT). EMT of tubular epithelial cells is the mechanism by which renal fibroblasts are generated. Here we show that miR-200 family members inhibit transforming growth factor-beta (TGF-beta)-induced EMT of tubular cells. Unilateral ureter obstruction (UUO) is a common model of EMT of tubular cells and subsequent tubulointerstitial fibrosis. In order to examine the role of miR-200 family members in tubulointerstitial fibrosis, their expression was investigated in the kidneys of UUO mice. The expression of miR-200 family miRNAs was increased in a time-dependent manner, with induction of miR-200b most pronounced. To clarify the effect of miR-200b on tubulointerstitial fibrosis, we injected miR-200b precursor intravenously. A single injection of 0.5 nM miR-200b precursor was sufficient to inhibit the increase of collagen types I, III and fibronectin in obstructed kidneys, and amelioration of fibrosis was confirmed by observation of the kidneys with Azan staining. miR-200 family members have been previously shown to inhibit EMT by reducing the expression of ZEB-1 and ZEB-2 which are known repressors of E-cadherin. We demonstrated that expression of ZEB-1 and ZEB-2 was increased after ureter obstruction and that administration of the miR-200b precursor reversed this effect. In summary, these results indicate that miR-200 family is up-regulated after ureter obstruction, miR-200b being strongly induced, and that miR-200b ameliorates tubulointerstitial fibrosis in obstructed kidneys. We suggest that members of the miR-200 family, and miR-200b specifically, might constitute novel therapeutic targets in kidney disease.